Western Diet Decreases Hepatic Drug Metabolism in Male LDLr−/−ApoB100/100 Mice

Consumption of a Western diet is an important risk factor for several chronic diseases including nonalcoholic fatty liver disease (NAFLD), but its effect on the xenobiotic metabolizing enzyme activities in the liver has been studied incompletely. In this study, male LDLr−/−ApoB100/100 mice were fed with Western diet (WD) or a standard diet for five months to reveal the effects on drug metabolism such as cytochrome P450 (CYP) oxidation and conjugation activities in the liver. Hepatic steatosis, lobular inflammation, and early fibrosis were observed in WD fed mice, but not in chow diet control mice. When compared to the controls, the WD-fed mice had significantly decreased protein-normalized CYP probe activities of 7-ethoxyresorufinO-deethylation (52%), coumarin 7-hydroxylation (26%), 7-hydroxylation of 3-(3-fluoro-4-hydroxyphenyl)-6-methoxycoumarin (70%), 7-hydroxylation of 3-(4-trifluoromethoxyphenyl)-6-methoxycoumarin (78%), 7-hydroxylation of 3-(3-methoxyphenyl)coumarin (81%), and pentoxyresorufin O-depentylation (66%). Increased activity was seen significantly in sulfonation of 3-(4-methylphenyl)-7-hydroxycoumarin (289%) and cytosol catechol O-methyltranferase (COMT, 148%) in the WD group when compared to the controls. In conclusion, the WD-induced steatosis in male LDLr−/−ApoB100/100 mice was associated with decreased CYP oxidation reactions but had no clear effects on conjugation reactions of glucuronidation, sulfonation, and cytosolic catechol O-methylation. Consequently, the WD may decrease the metabolic elimination of drugs compared to healthier low-fat diets.


Introduction
Consumption of the so-called Western diet (WD) is a key risk factor for several chronic diseases such as type 2 diabetes, obesity, coronary heart diseases, and degenerative diseases [1]. Te WD also promotes the development of several type of liver disorders from the benign form of nonalcoholic fatty liver disease (NAFLD) to the more severe nonalcoholic steatohepatitis (NASH) with or without fbrosis [2]. In NAFLD, an excessive amount of triglycerides accumulate in hepatocytes, leading to hepatic steatosis and chronic infammation, degeneration of hepatocytes, necrosis, and the formation of fbrosis [2,3].
Te liver is the most important organ for the metabolism of xenobiotics, e.g., drugs, whose efects and efective time are greatly afected by their metabolism in the liver [4,5]. Te metabolism of drugs and other xenobiotics occurs in oxidation, reduction, hydrolysis, and conjugation reactions catalyzed by drug-metabolizing enzymes. Te most important group of the drug-metabolizing enzymes are cytochrome P450s (CYPs), which are responsible for the metabolism of about 75% of clinically used drugs [6,7]. Tese reactions are catalyzed mainly by fve liver CYP enzymes, i.e., CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Some other important xenobiotic-metabolizing enzymes are UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), which can conjugate the hydroxylated metabolites of CYP oxidation reactions. Both the types of drug-metabolizing enzymes and their amounts in the liver determine the rate and routes of metabolism and thus the elimination of xenobiotics. Te same types of reactions take place for xenobiotics in humans and other mammalian species by the corresponding drugmetabolizing enzymes, although there are variations of levels, and the primary structures of the metabolizing enzymes often cause diferences in rates and pathways between species [8].
While the basic level of drug metabolism and enzymes involved such as CYPs, UGTs, and SULTs is determined by the genome and age of the individual, they are also afected by several exogenous factors such as health-disease status, the presence of obesity, the diet, as well as exposure to inhibiting or inducing substances [4,5]. In obese individuals, CYP2E1, 2D6, 3A4, and 3A5 metabolized drugs are eliminated faster [9][10][11]. Conversely, CYP1A2 has been observed to be suppressed in obese and NAFLD patients, and fnally those drugs which are dependent on CYP1A2 oxidation such as theophylline are eliminated slowly in these individuals [12]. In animal models, CYP2C and CYP2E have been associated with faster xenobiotic metabolism in obese animals, whereas other liver CYP displayed signs of decreased metabolism [13]. Te changes in conjugation reactions in NAFLD patients seem to be dependent on the type of conjugation, e.g., activity of SULT2A1 is decreased [14], whereas changes in glucuronidation rates have been inconsistent [14][15][16].
Mice with genetic manipulations such as leptin defciency (ob/ob) [17], dysfunctional leptin receptor (db/db), or disturbances in lipid metabolism such as defciency in low-density lipoprotein receptor (LDLr −/− ) and expression of apolipoproteins or their combinations are used as models to mimic metabolic disorders [18,19]. LDLr-defcient mice expressing only apolipoprotein B100 (LDLr −/− ApoB 100/100 ) are a model in which spontaneous mild hypercholesterolemia and hypertriglyceridemia exist in conjunction with an increased plasma level of LDL [19,20]. Terefore, we anticipated that these mice would be likely to develop moderate hepatic steatosis after prolonged consumption of a WD, and this would be accompanied by obesity.
Te aim of the study was to investigate whether consumption of a WD would afect the xenobiotic-metabolizing enzyme activities in the liver of male LDLr −/− ApoB 100/100 mice. Terefore, probe activities of CYP, UGT, SULT, and catechol O-methyltransferase (COMT) of microsomal or cytosol samples were determined from the liver samples of male LDLr −/− ApoB 100/100 mice fed with the WD or the standard diet for fve months.

Mice.
Tree-months-old male LDLr −/− ApoB 100/100 mice from the colony of the Center of Experimental Animals at the University of Eastern Finland (129sv/B6 mixed background, backcrossed ten times, Te Jackson Laboratory, Bar Harbor, ME, USA) were randomly divided into two groups, a Western diet (WD) (n � 10) and regular chow diet (n � 8) groups. Te WD group was fed with the WD (Harlan Teklad 88137, containing 42% of kcal from fat), and control mice received a standard diet (Teklad Global 16% protein rodent diet: 12% of calories from fat and 0% cholesterol) for fve months. Mice were euthanized with CO 2 and perfused with phosphate-bufered saline to collect liver samples for histology or snap frozen in liquid nitrogen. Mice were housed in groups with free access to tap water in a room with a controlled 55% humidity and a temperature of 22°C. A 12 h light and 12 h dark environmental light cycle was maintained.
Te experimental design was approved by the Animal Ethics Committee of the State Provincial Ofce of Southern Finland (Decision number ESAVI/11642/04.10.07/2014). All the experiments conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientifc purposes and by the ARRIVE guidelines.

Preparation of Microsomes.
Cytosol and microsome specimens were prepared from frozen livers as described earlier [26]. Shortly, the liver tissue samples (left and right lobes of the liver) were randomized, weighed, gently thawed, and homogenized with Potter-Elvehjem homogenizer in 100 mM Tris-HCl bufer pH 7.4 containing 1 mM EDTA (4 parts bufer to 1 part tissue). Te homogenate was centrifuged at 10 000 g for 15 min at 4°C. Te supernatant was collected and centrifuged at 100 000 g for 60 min at 4°C. Te supernatant and pellet were separated. Te pelleted microsomal fraction was rehomogenized with Potter-Elvehjem homogenizer in 100 mM Tris-HCl bufer, pH 7.4, containing 0.1 mM EDTA and 20% glycerol. Te protein concentration was determined with the Bradford method of Bio-Rad protein. Te supernatant and microsomal samples were stored at −80°C until further use.
Measurements (both CYP and conjugation) were carried out in a randomized order of samples at 37°C in a 96 multiwell plate format following fuorescence every other minute for 40 min using an excitation flter at 405 nm and detection at 460 nm for oxidation of coumarin and coumarin derivatives, and at excitation 570 nm and emission 615 nm for 7-ethoxyresorufn or 7-pentoxyresorufnO-dealkylations in a Victor 2 1420 Multilabel counter (PerkinElmer, Life Sciences, Turku, Finland). Resorufn was used as a standard for 7-Odealkylation of resorufns and 7-hydroxycoumarin as the surrogate standards for oxidation of coumarin or coumarin derivatives to calculate the amount of product formed. Te linear phase of the reactions was used for calculations of CYP activities.

Statistical
Analysis. An unpaired parametric t-test with two-sidedp values was carried out on data of all 14 enzyme activities using GraphPad Prism 5 software. For multivariate analysis, SIMCA 15.0. Umetrics software was used when conducting the principal component analysis (PCA) to investigate latent components explaining the variation in the data and partial least squares discriminant analysis (PLS-DA) to determine latent components explaining the separation between the study groups. Numerical values are shown as geometrical mean ± 95% confdence interval (CI).

Prolonged WD Induces Hepatic Steatosis and Fibrosis in
Male LDLr −/− ApoB 100/100 Mice. To induce hepatic steatosis, male LDLr −/− ApoB 100/100 mice were fed with the WD for fve months, whereas the control mice received the standard chow diet. Before the diet, the body weight was on average 20 g. After the chow diet, average weight was 30 g, and after the WD, average weight was 40 g. Te WD induced a clear increase in hepatic lipid accumulation, but on a chow diet, hepatic steatosis was not detected (Figures 1(a) and 1(b)). In addition, the WD increased the infltration of infammatory cells, such as neutrophils and lymphocytes, and promoted the formation of early fbrosis (Figure 1(d)), which was not detected on a chow diet in male LDLr −/− ApoB 100/100 mice after 5 months (Figure 1(c)).

Te Efect of WD on Liver Drug Metabolic Activities.
Seven CYP, two UGT, two SULT, and two COMT probe activities were measured from liver microsomes or cytosol of both chow diet and WD male LDLr −/− ApoB 100/100 mice. Te activity was calculated per amount of protein and per liver, as accumulation of fat could afect protein concentration and liver size. Consequently, activity per liver indicated total liver metabolic capacity. Te consumption of the WD for fve months decreased signifcantly six microsomal CYP probe activities and had no signifcant efect on the 3-(3-benzyloxo)phenyl-7-methoxycoumarin 7-O-demethylation rate in both the protein-normalized and per liver activity analysis ( Figure 2 and Table 1). In protein-normalized analysis, the WD decreased most extensively in the coumarin 7hydroxylation activity by decreasing it to 26% from the rate of chow diet mice. Other protein-normalized activities were decreased less, varying from 52% (7-ethoxyresorufn 7-O-deethylation) to 81% (7-hydroxylation of 3-(3-methoxyphenyl)coumarin) compared to the rates of chow diet mice. In the rates of glucuronidation, sulfonation, and microsomal COMT, there were no diferences in the protein-normalized activities between samples of the chow diet and WD (Figure 3), but activities of both glucuronidation and sulfonation of 4-trifuoromethyl-7-hydroxycoumarin and microsomal COMT per liver were decreased signifcantly (Table 1). Protein-normalized cytosolic COMT activity was 48% higher in WD mice compared to chow diet mice (Figure 3), but the per liver activity diference was not statistically signifcant (Table 1). In male LDLr −/− ApoB 100/100 mice, the WD afected more signifcantly on CYP probe activities than conjugation probe activities. Primarily, the WD decreased the CYP enzyme probe activities.

Discussion
Te liver is an important organ for kinetics of drugs and other xenobiotics because all compounds absorbed from the gastrointestinal tract have to pass through the liver before gaining access to the systematic circulation and further distribution in the body. Secondly, of all organs, the liver has the highest ability and capability to metabolize xenobiotics [4,5]. High-fat diets, such as WD, alters the function of the liver and afects hepatic xenobiotic metabolism [31]. In this study, the efects of the WD feeding on hepatic xenobiotic metabolism were studied in genetically modifed hypercholesterolemic LDLr −/− ApoB 100/100 male mice. Prolonged WD consumption induced severe lipid accumulation, chronic infammation, and early fbrosis in male LDLr −/− ApoB 100/100 mice liver. Mice fed with a WD had lower hepatic activities of several drug-metabolizing enzymes than mice fed with the chow diet. Te declines of CYP enzyme activities were statistically signifcant, whereas the decreases in conjugation activities of glucuronidation and sulfonation were not statistically signifcant. Te chow diet and WD mice were observed to difer metabolically, as multivariate PLC-DA analysis separated them to higher metabolism control and lower metabolism WD groups. According to these results, WD-induced hepatic steatosis decreased the overall liver xenobiotic metabolism of male LDLr −/− ApoB 100/100 mice.
Te efects of WD on the oxidation rates of seven probe substrates of CYP enzymes were determined in this study. Ethoxyresorufn 7-O-deethylation [32], coumarin 7hydroxylation [28], and pentoxyresorufn 7-Odepentylation [33] are identifed as the probe substrates of Table 1: Te efect of a Western diet (WD) on the total probe drug metabolism of the mouse model of NAFLD. Te liver microsome and cytosol fractions were prepared from the livers of eight months old male LDLr −/− ApoB 100/100 mice that were fed either with chow (8 mice) or WD (10 mice) for fve months.
In contrast, high-fat diets have been observed to increase liver CYP activities in several animal models. In A/J and wild-type mice, a high-fat diet increased both hepatic CYP1A2 [34] and 2E1 activity, respectively, compared to a low-fat diet [35]. CYP2E1 is involved in the metabolism of ketogenic substances [36]. Furthermore, a high-fat diet has increased signifcantly CYP2A5 enzyme activity in C57BL/6J mice [37]. A high-fat diet has been shown to signifcantly increase CYP1A2, 2E1, 2C, and 4A enzyme activities in streptozotocin-induced diabetes in Sprague-Dawley rats [38]. In male domestic pigs, the continuous consumption of the high-fat diet increased the CYP2E1 activity but did not alter 1A or 2B enzyme activity [39]. In C57BL/6 mice, the WD has not altered liver mRNA levels of xenobiotic metabolizing CYPs, but instead has decreased mRNA levels of CYP7 and CYP51 [40], which are bile acid and steroid synthetizing CYPs, respectively. Single-or seven-day daily i.p. exposure of oil has reduced the amounts of CYP enzyme proteins and activities in the mice liver [41]. Alternatively, in rats, the lipid exposure has increased the hepatic activity and protein level of CYP2E [42,43]. In Sprague-Dawley rats, a high-fat diet has exerted no signifcant efect on CYP2B1 mRNA or protein expression levels [44]. In two of these studies, the high-fat diets have resulted in hepatic steatosis and infammation in C57BL/6J mice liver histology [35,37]. In summary, it is evident that the efect of consuming highfat diet on liver CYP enzyme activities is dependent on the duration and amount of fat in diet and severity of the efect on the liver histology. Our study has some limitations. We did not monitor consumption of the food or caloric intake. Te weight of the mice was measured only before and after WD. We did not include wild-type mice in this study. However, LDLr −/− ApoB 100/100 mice is a model of human familial hypercholesterolemia, so these results may have implications in individuals with hypercholesterolemia. In addition, the experiments were performed only on male mice, which may afect the generalizability of the results.
Journal of Nutrition and Metabolism 9 [46]. Tere was no data found on the efects of a high-fat diet on SULTs or cytosolic COMT.

Conclusions
Te efects of WD on the liver drug metabolism in male LDLr −/− ApoB 100/100 mice were studied here for the frst time. Te fve-month consumption of the WD-induced hepatic steatosis in male LDLr −/− ApoB 100/100 mice. CYP1, CYP2A, and CYP2B probe activities were signifcantly lower in mice fed with the WD than in mice who were fed with a standard chow diet. No clear efects were observed on conjugation reactions of glucuronidation, sulfonation, and catechol O-methylation after feeding with a WD. Multivariate analysis from hepatic drug metabolizing probe activities separated WD mice into a low xenobiotic metabolism activity group and the chow diet mice as the high xenobiotic metabolism activity group. In conclusion, WD-induced obesity and hepatic steatosis were associated with decreased liver xenobiotic metabolism in male LDLr −/− ApoB 100/100 mice.

Data Availability
Te analyzed data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest. Table 2: Linear correlation coefcients between oxidation rates of two CYP substrates or between rates of two conjugation substrates and their respective P values in liver samples of male LDLr −/− ApoB 100/100 mice fed with the chow or Western diet. Te rates of CYP oxidation, glucuronidation, sulfonation, or COMT with the indicated substrates were determined as described in Materials and Methods. Correlations were calculated from the data in Figures 2 and 3.